Adipose-Derived Mesenchymal Stem Cells Prevent Systemic Loss in Collagen-Induced Arthritis

This information is current as Manasa G. Garimella, Supinder Kour, Vikrant Piprode, of September 27, 2021. Monika Mittal, Anil Kumar, Lekha Rani, Satish T. Pote, Gyan C. Mishra, Naibedya Chattopadhyay and Mohan R. Wani J Immunol 2015; 195:5136-5148; Prepublished online 4

November 2015; Downloaded from doi: 10.4049/jimmunol.1500332 http://www.jimmunol.org/content/195/11/5136

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Adipose-Derived Mesenchymal Stem Cells Prevent Systemic Bone Loss in Collagen-Induced Arthritis

Manasa G. Garimella,* Supinder Kour,* Vikrant Piprode,* Monika Mittal,† Anil Kumar,* Lekha Rani,* Satish T. Pote,* Gyan C. Mishra,* Naibedya Chattopadhyay,† and Mohan R. Wani*

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammatory synovitis leading to joint destruction and systemic bone loss. The inflammation-induced bone loss is mediated by increased osteoclast formation and function. Current antirheumatic therapies primarily target suppression of inflammatory cascade with limited or no success in controlling progres- sion of bone destruction. Mesenchymal stem cells (MSCs) by virtue of their tissue repair and immunomodulatory properties have shown promising results in various autoimmune and degenerative diseases. However, the role of MSCs in prevention of bone destruction in RA is not yet understood. In this study, we investigated the effect of adipose-derived MSCs (ASCs) on in vitro Downloaded from formation of bone-resorbing osteoclasts and pathological bone loss in the mouse collagen-induced arthritis (CIA) model of RA. We observed that ASCs significantly inhibited receptor activator of NF-kB ligand (RANKL)–induced osteoclastogenesis in both a contact-dependent and -independent manner. Additionally, ASCs inhibited RANKL-induced osteoclastogenesis in the presence of proinflammatory cytokines such as TNF-a, IL-17, and IL-1b. Furthermore, treatment with ASCs at the onset of CIA significantly reduced clinical symptoms and joint pathology. Interestingly, ASCs protected periarticular and systemic bone loss in CIA mice by maintaining trabecular bone structure. We further observed that treatment with ASCs reduced osteoclast precursors in bone http://www.jimmunol.org/ marrow, resulting in decreased osteoclastogenesis. Moreover, ASCs suppressed autoimmune T cell responses and increased the percentages of peripheral regulatory T and B cells. Thus, we provide strong evidence that ASCs ameliorate inflammation-induced systemic bone loss in CIA mice by reducing osteoclast precursors and promoting immune tolerance. The Journal of Immunology, 2015, 195: 5136–5148.

uman (RA) is an autoimmune disease myeloid precursors of monocyte/macrophage lineage in the pres- characterized by chronic inflammatory synovitis and pro- ence of receptor activator of NF-kBligand(RANKL)andM-CSF H duction of autoantibodies and several proinflammatory (2). The proinflammatory milieu of the arthritic synovium leads to by guest on September 27, 2021 cytokines, which together lead to joint destruction (1). Inflammation- aberrant differentiation and activation of osteoclasts, resulting in induced bone destruction is a key pathological feature of RA, massive bone loss (3). Chronic inflammation also shifts the equi- mediated by osteoclasts. Osteoclasts are formed by the fusion of librium of bone remodeling toward increased bone resorption rather than formation, leading subsequently to systemic osteopenia asso- ciated with an enhanced risk for osteoporotic fractures (4). Current *National Centre for Cell Science, Ganeshkhind, Pune 411 007, India; and †Division of Endocrinology, Council of Scientific and Industrial Research–Central antirheumatic therapies primarily target the suppression of inflam- Drug Research Institute, Lucknow 226 031, India matory cascade with varying success in limiting the progression of Received for publication February 10, 2015. Accepted for publication September 30, bone destruction (5). Therefore, better therapeutic agents are needed 2015. affecting both inflammatory processes and skeletal damage. This work was supported by Department of Biotechnology, India Grant BT/HRD/34/ Mesenchymal stem cells (MSCs) are adult multipotent non- 01/2009 (to M.R.W.), as well as Council of Scientific and Industrial Research, New hematopoietic stem cells of mesodermal origin that can differen- Delhi, India Grant BSC0201 (to N.C.). M.G.G. is the recipient of a senior research fellowship from the Council for Scientific and Industrial Research, New Delhi, India. tiate into cells of both mesenchymal and nonmesenchymal lineages Address correspondence and reprint requests to Dr. Mohan R. Wani, National Centre (6, 7). Initially identified from bone marrow, MSCs have been iso- for Cell Science, Savitribai Phule Pune University Campus, Ganeshkhind Road, Pune lated from various sources, including adipose tissue, gingiva, um- 411 007, India. E-mail address: [email protected] bilical cord, and many other adult tissues (8). In addition to their The online version of this article contains supplemental material. regenerative potential, they exert profound immunomodulation by Abbreviations used in this article: ASC, adipose-derived mesenchymal stem cell; affecting proliferation, differentiation, and maturation of various im- BMD, bone mineral density; Breg, regulatory B cell; BV/TV, trabecular bone volume fraction; CFU-F, CFU-fibroblast; CIA, collagen-induced arthritis; CII, collagen type mune cell types (9). MSCs are also known to home to the site of II; CM, conditioned medium; Conn. D, connectivity density; Cs. Th, cross-sectional injury or inflammation and contribute to tissue repair processes lo- thickness; mCT, microcomputed tomography; Ct. BMD, cortical BMD; CTR, calci- tonin receptor; DC-STAMP, dendritic cell–specific transmembrane protein; dLN, cally through trophic factors (10). Because of the unique combination draining lymph node; MMP, matrix metalloproteinase; MNC, multinuclear cell; of these properties, MSCs are being widely studied in clinical trials of MSC, mesenchymal stem cell; OCP, osteoclast precursor; RA, rheumatoid arthritis; various autoimmune disorders and degenerative diseases (11, 12). RANK, receptor activator of NF-kB; RANKL, receptor activator of NF-kB ligand; rm, recombinant murine; Tb. BMD, trabecular BMD; Tb. N, trabecular number; Despite several conflicting reports, MSCs from various tissues Tb. Sp, trabecular separation; Tb. Th, trabecular thickness; TRAP, tartrate-resistant have shown protective effect in reducing autoimmune and inflam- acid phosphatase; Treg, regulatory T cell. matory processes in preclinical models of RA (13–15). However, This article is distributed under The American Association of Immunologists, Inc., the role of MSCs in protecting skeletal component of arthritis, in- Reuse Terms and Conditions for Author Choice articles. cluding pathological bone loss and the bone-resorbing osteoclasts, Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 has not been well studied. Also, earlier studies on the role of MSCs www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500332 The Journal of Immunology 5137 in osteoclast differentiation are conflicting, with both stimulatory cultured in a 60-mm petri dish for 7–10 d followed by formalin fixation (16, 17) as well as inhibitory (18, 19) effects. Therefore, in the and staining with hematoxylin. Cultures were observed for colonies with present study, we investigated the effect of MSCs on the differen- aggregates of 50 or more cells using a bright-field microscope and were scored as CFU-F. tiation of bone-resorbing osteoclasts and inflammation-induced bone loss in the mouse collagen-induced arthritis (CIA) model. In vitro osteogenic, adipogenic, and chondrogenic We used syngeneic adipose-derived MSCs (ASCs) for our study differentiation of ASCs because of their ease of isolation and relative abundance. Induction of osteoblast, adipocyte, and chondrocyte differentiation was In this study, we found that ASCs inhibited RANKL-induced performed as described previously (21). For osteoblast differentiation, 3 2 osteoclast differentiation in vitro in both a contact-dependent and monolayer cultures of mouse ASCs (2–5 3 10 cells/cm ) were incubated a b m -independent manner. ASCs protected against disease severity and in -MEM containing 10% FCS, 10 mM -glycerophosphate, 50 g/ml ascorbic acid, and 100 nM dexamethasone for 21–25 d. Cultures were half bone loss in CIA mice as evaluated by histomorphometric measure- fed every third day and assessed for matrix mineralization by Alizarin red ments of trabecular bone indices. Decreased bone loss was found to be S staining. For Alizarin red S staining, cells were washed with PBS, fixed a result of decreased osteoclast precursors (OCPs) in bone marrow and in 10% formalin, and incubated with 2% Alizarin red S for 2 min at room increased peripheral regulatory T cell (Treg) and B cell (Breg) per- temperature. Cells were washed with distilled water and observed for mineralization as red calcium deposits. Alizarin red S was eluted in 10% centages. This study clearly suggests that ASCs exert a strong ther- glacial acetic acid and quantitated colorimetrically at 405 nm. apeutic effect in RA by preventing bone loss along with dampening the For adipogenic differentiation, ASCs (2–5 3 103 cells/cm2) were in- ongoing inflammatory responses. cubated until they attained 90% confluency. Adipogenesis was induced using a commercial kit from Lonza. Briefly, cells were incubated in adi- pogenic induction medium (DMEM with 4.5 g/l glucose, 10% FCS, 1 mM Downloaded from Materials and Methods dexamethasone, 10 mM insulin, 0.5 mM 3-isobutyl-1-methylxanthine, and Animals 100 nM indomethacin) for 3 d, followed by 1 d in maintenance medium m Male and female DBA/1J (6–12 wk old) mice were obtained from the (DMEM with 4.5 g/l glucose, 10% FCS, and 10 M insulin) constituting Experimental Animal Facility of the National Centre for Cell Science, one cycle of induction and maintenance. After multiple rounds of induc- Pune, India. Water and food were provided ad libitum. All experiments tion and maintenance for 25–30 d, cells were examined for formation of oil involving animal use were approved by the Institutional Animal Ethics globules by Oil Red O staining. Cells were formalin fixed, stained with Oil Committee. Red O (3 mg/ml) for 1 h at room temperature, and were observed under a bright-field microscope for the formation of oil globules. http://www.jimmunol.org/ Abs and reagents For chondrogenic differentiation, 2 3 105 ASCs were pelleted down in round-bottom 96-well plates to form a pelleted micromass at the bottom of ε The following Abs were purchased from BD Biosciences: purified anti-CD3 the well. Pellets were incubated in serum-free DMEM (4.5 g/l glucose) (145-2C11, NA/LE), purified anti-CD28 (37.51, NA/LE), Pacific Blue (PB)– containing 0.1 mM dexamethasone, 50 mg/ml ascorbic acid, 1 mM sodium anti-CD4 (RM4-5), allophycocyanin–anti-Foxp3 (FJK-16s), PE–anti-CD19 pyruvate, 40 mg/ml L-proline, 6.25 mg/ml ITS, and 10 ng/ml TGF-b3. (1D3), Alexa Fluor 488–anti-c-fms (AFS98), PE-Cy7–anti-CD11b (M1/70), After 21 d, cell pellets were fixed in 10% formalin, and 5-mm sections allophycocyanin–anti-CD11c (HL3), allophycocyanin–anti-F4/80 (BM8), were stained with H&E to evaluate histology. PE–anti-RANK (R12-31), PE–anti-c-Kit (2B8), PE–anti-B220 (RA3-6B2), PerCP-Cy5.5–anti-CD1d (1B1), PE-Cy7–anti-CD5 (53-7.3), PE–anti- Preparation of conditioned medium from ASCs CD105 (MJ7/18), allophycocyanin–CD29 (HMb1-1), FITC–anti-CD44

ASC-conditioned medium (ASC-CM) was prepared by culturing ASCs of by guest on September 27, 2021 (IM7), FITC–anti-Sca-1 (D7), FITC–anti-MHC class II, PE–anti-CD90 a (53-2.1), FITC–anti-CD34 (RAM34), FITC–anti-CD14 (rmC5-3), allophy- passages 2 or 3 at 70–90% confluency in -MEM supplemented with 10% FCS for 24 h. Culture supernatant was collected, centrifuged, and stored at cocyanin–anti-platelet-derived growth factor receptor (APA5), PE–anti-H- 2 2Dd (34-5-8S), PE–rat IgG2ak, PerCP-Cy5.5–rat IgG2bk, PE–rat IgG2bk, 80˚C until use. PE–Cy7-rat IgG2bk, allophycocyanin–rat IgG2ak, Alexa Fluor 488–rat Induction of CIA IgG2ak, Pacific Blue–rat IgG2bk, allophycocyanin–armenian hamster IgG1l2, PE-Cy7–rat IgG2b,k, PE-Cy7–rat IgG2ak, PE-Cy7–rat IgG1, Native CII was dissolved in 50 mM acetic acid at 4˚C overnight and allophycocyanin–armenian hamster IgG, FITC–rat IgG2bk, FITC–rat emulsified with an equal volume of CFA (containing 4 mg/ml Mycobacte- IgG2ak, FITC–rat IgG1k, PE–mouse IgG2ak. PE-Cy7–anti-CD73 (TY/11.8) rium tuberculosis; strain H37Ra). DBA/1J mice (8–10 wk old) were injected and PE–anti-IL-17A (eBio17B7) were from eBioscience. with 100 ml emulsion containing 200 mg CII intradermally at the base of the Foxp3 staining buffer and RBC lysis buffer were from eBioscience. tail. At day 21 after primary immunization, mice were given a booster with Mouse T cell activation Dynabeads were obtained from Life Technologies. 200 mg CII emulsified in an equal volume of IFA, through the same route, PMA and ionomycin, dexamethasone, b-glycerophosphate, ascorbic acid, and randomly divided into two groups. On day 22, 2 3 106 murine syn- naphthol AS-BI phosphate, sodium tartarate, native chicken sternal col- geneic ASCs in 100 ml PBS per mouse were administered i.p. to one group lagen type II (CII), and IFA were purchased from Sigma-Aldrich. CFA was (CIA plus ASCs) and an equal volume of PBS was administered to the other from Chondrex. Recombinant human M-CSF, recombinant murine (rm) group (CIA plus PBS). Mice were monitored for signs of arthritis by two TNF-a, rmIL-17, rmIL-6. and rmIL-1b were obtained from R&D Systems. blinded observers using two clinical parameters: paw swelling and arthritic Soluble rmRANKL was from PeproTech. Adipogenic and chondrogenic score. Paw swelling was evaluated by measuring thickness of affected induction kits were obtained from Lonza. All cultures were maintained at hindpaws using 0–10 mm calipers. Clinical arthritis was assessed by the 37˚C in a humidified atmosphere of 5% CO2 in air. following scoring system: 0, no evidence of erythema and swelling; 1, er- ythema and mild swelling limited to tarsals or ankle joint; 2, erythema and Isolation of ASCs mild swelling extending from ankle to tarsal joints; 3, erythema and mod- Murine ASCs were isolated as described by Sung et al. (20) with slight erate swelling extending from ankle to metatarsal joints; 4, erythema and modifications. Subcutaneous adipose tissue isolated from 10- to 12-wk-old severe swelling surrounding the ankle, foot, and digits. Each limb was DBA/1J mice was digested with 2 mg/ml collagenase (type 1A, Sigma- graded individually, giving a maximum possible score of 16 per animal. Aldrich) in PBS at 37˚C for 15–20 min. The cell suspension obtained after Hindlimbs were evaluated by soft x-ray radiography (Siemens). Mice were filtration through 40-nm nylon filter mesh (BD Falcon) was centrifuged sacrificed on day 36 and studied for various disease parameters. 4 and then seeded at a density of 5 3 10 cells/ml and cultured in DMEM Histology (containing 4.5 g/l glucose) supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Nonadherent For histological analysis, whole knee joints were excised and fixed in 10% cells were discarded after 72 h and adherent cells were maintained until formalin for 4 d. Decalcification was done in 5% formic acid and the they attained 80–90% confluency. Cells were passaged using trypsin- specimens were processed for paraffin embedding. Next, 5-mm sections EDTA and split in a ratio of 1:3. were prepared and stained with H&E. CFU-fibroblast assay Measurement of serum anti-CII Abs To assess the clonogenic potential of ASCs, a CFU-fibroblast (CFU-F) assay Peripheral blood was collected from mice by orbital sinus puncture and sera was performed as described previously (21). Briefly, 1 3 103 cells were were obtained using BD vaccutainer tubes. Sera were then tested for anti-CII 5138 STEM CELL THERAPY IN MOUSE MODEL OF RHEUMATOID ARTHRITIS

Abs by ELISA using a commercial kit (Chondrex) following the manu- population and measured for CD1dhi and CD5+ populations. All gates were facturer’s instructions. set based on isotype controls. T cell proliferation assay Osteoclastogenesis and tartrate-resistant acid phosphatase Single-cell suspensions were prepared from draining lymph nodes (dLNs) and measurement 5 cultured in 96-well plates (5 3 10 cells/well) in presence of CII (40 mg/ml) Murine osteoclasts were generated as described previously (22). Briefly, or plate-bound anti-CD3ε (2 mg/ml) for 72 h. Cells were pulsed with 3 bone marrow cells from mice were cultured overnight with 7 ng/ml [ H]thymidine (1 mCi/well) during the last 18 h of the culture period recombinant human M-CSF. Nonadherent cells were harvested, RBC and harvested using a 96-well cell harvester (PerkinElmer). Incorporated lysed, and cultured in the presence of M-CSF (30 ng/ml) and varying radioactivity was measured as cpm, using a beta liquid scintillation counter concentrations of soluble RANKL for 3–4 d. Tartrate-resistant acid (Packard). For in vitro coculture assays of ASCs and T cells, CD4+ T cells + phosphatase (TRAP) staining was then performed in presence of 0.05 M were isolated from spleens of normal mice using CD4 T cell enrichment sodium tartrate using napthol AS-BI phosphate as substrate. TRAP+ cells mixture (BD Biosciences) by magnetic selection. Stimulation index was with three or more nuclei were counted as osteoclasts. calculated by the formula: cpm in response to Ag/cpm in absence of Ag. Pit formation assay Flow cytometric analysis Osteoclasts were differentiated as mentioned above in 96-well plates coated Single-cell suspensions prepared from peritoneum, synovium, peripheral with a thin film of calcium phosphate (Osteo assay plates, Nunc) for 8 d. blood, spleen, and dLNs were stained with Pacific Blue–anti-CD4 Ab for Cells were bleached with 4% sodium hypochlorite for 2 min and resorption 30 min. Cells were washed thoroughly with stain buffer (Dulbecco’s PBS pits were observed under a bright-field microscope as clear zones in the plus 2% FCS) and fixed/permeabilized with Foxp3 permeabilization buffer matrix. Percentage resorption was calculated by considering the area of for 60 min. Intracellular staining was performed using allophycocyanin- single well as 100% with six to eight wells per group. conjugated anti-Foxp3 Ab in permeabilization buffer for 45 min. Cells Downloaded from were washed with permeabilization buffer and analyzed by flow cytometry. RNA extraction and analysis by RT-PCR For surface staining, single-cell suspensions of ASCs, splenocytes, or bone marrow cells were fixed with 4% paraformaldehyde for 10 min at room RNA was isolated using TRIzol reagent (Invitrogen) and total RNA (2 mg) temperature and washed with stain buffer. Then, 1 3 105–106 cells per tube was used for synthesis of cDNA by reverse transcription (cDNA synthesis kit; were stained with fluorochrome-conjugated Abs for 45 min on ice, along Invitrogen). cDNA were used for real-time PCR using SYBR Green master with appropriate isotype controls in separate tubes. Cells were washed with mix (Applied Biosystems) for 40 cycles on a StepOnePlus system (Applied stain buffer twice and acquired on a FACSCanto II (BD Biosciences). All Biosystems). Each cycle consisted of 30 s denaturation at 94˚C, 30 s http://www.jimmunol.org/ steps were performed in dark at 4˚C. Data were analyzed using FlowJo annealing at 58–60˚C, and 30 s extension at 72˚C, followed by a melt curve at (Tree Star) software. the end. GAPDH was used as endogenous control. The primer sequences For Tregs, lymphocytes based on forward scatter and sides scatter were used were: TRAP (forward, 59-GGAACTTCCCCAGCCCTTAC-39,reverse, gated and measured for percentages of CD4 and Foxp3 dual-positive 59-AGGTCTCGAGGCATTTTGGG-39), integrin b3 (forward, 59-AGAATGC populations. For Bregs, a lymphocyte cluster was gated for the CD19+ CTGCTTGCCCATGT-39,reverse,59-TACGGGACACGCTCTGTTTCT-39), by guest on September 27, 2021

FIGURE 1. Culture-expanded murine ASCs exhibit multilineage differentiation and immunosuppressive potential. ASCs were isolated from s.c. adipose tissue of DBA/1J mice and culture expanded as described in Materials and Methods.(A) Cells of passage 2 were analyzed for their clonogenic potential by CFU-F assay. (B) Surface phenotyping for the expression of mesenchymal and hematopoietic markers by flow cytometry. Numbers indicate percentage expression of the marker with respect to their isotype controls. (C–E) Osteogenic, adipogenic, and chondrogenic differentiation of ASCs was induced in respective differentiation media. Matrix mineralization by differentiated osteoblasts was assessed by Alizarin red S staining. Adipocytes were characterized by Oil Red O staining. Chondrogenic differentiation shows cells in lacunae in H&E-stained sections of micromass pellet cultures (original magnification 310). (F) Suppression of activated CD4+ T cell proliferation by ASCs or (G) ASC-CM was assessed by coculturing splenic CD4+ Tcells(2.53 105 cells/ml) with ASCs (2.5 3 103 cells/ml) or ASC-CM (50% of culture volume) under activated or unactivated conditions for 72 h. T cell activation Dynabeads were used for activating T cells, and proliferation was measured by a [3H]thymidine incorporation assay. Bar graphs are expressed as mean 6 SEM of three to five replicates per group. ***p # 0.001. (A)–(E) are representative of more than five batches of ASCs. (F)and(G) are representative of two independent experiments. The Journal of Immunology 5139 calcitonin receptor (CTR; forward, 59-TGCCAACCATTATCCACGCCA-39, trabecular thickness (Tb. Th; mm), trabecular separation (Tb. Sp; mm), reverse, 59-TCACAAGCACGCGGACAATGT-39), dendritic cell–specific and connectivity density (Conn. D; 1/mm3). Cortical bone was also transmembrane protein (DC-STAMP; forward, 59-AGAGCTGTTGACTTCC analyzed for BMD (cortical BMD [Ct. BMD]) and cross-sectional GGG-39,reverse,59-ATACTTCCAGCCACAAGGGC-39), cathepsin K (for- thickness (Cs. Th; mm). ward, 59-TGCCTTCCAATACGTGCAGCA-39,reverse,59-TGCATTTAGCT GCCTTTGCCG-39), matrix metalloproteinase (MMP)9 (forward, 59-CTGT Statistical analysis 9 9 9 CGGCTGTGGTTCA-3 , reverse, 5 -AGACGACATAGACGGCAT-3 ), Results are expressed as mean 6 SEM or mean 6 SD for various ex- RANK (forward, 59-TTCGACTGGTTCACTGCTCC-39,reverse,59-CCTCA 9 9 perimental groups. The statistical differences were analyzed using an un- GAATCCACCGTGCTT-3 ), c-fms (forward, 5 -ACAAGGCAGGCTGGAA paired Student t test for comparison between two groups or by a one- or TAAT-39, reverse, 59-TGGGCTTCATCACACCTATC-39), and GAPDH two-way ANOVA followed by a Bonferroni correction for multiple com- (forward, 59-GGTGCTGAGTATGTCGTG-39,reverse,59-CCTTCCACATG parisons. A p value ,0.05 was considered statistically significant. CCAAAG-39). Analysis was done by the comparative CT method and fold change was calculated by the 22ΔΔCT method. Results Microcomputed tomography Culture-expanded murine ASCs exhibit multilineage Microcomputed tomography (mCT) of excised was carried out differentiation and immunosuppressive potential by a SkyScan 1076 CT scanner (SkySkan, Aartselaar, Belgium) as We used ASCs from DBA/1J mice for the present study because described previously for mice bone (23, 24) and following the general guidelines used for assessment of bone microarchitecture in rodents of their high proliferation rate and attainment of homogeneity in using mCT (25). Briefly, scanning was done at 50 kV, 200 mAusinga cultures at early passages. ASCs from initial passages (P2 or P3) 0.5-mm aluminum filter at a resolution of 9 mm/pixel. Reconstruction of were characterized for their clonogenicity, expression of MSC sections was achieved using a modified Feldkamp cone-beam algorithm markers, and multilineage differentiation. ASCs exhibited fibroblast- Downloaded from with beam hardening correction set to 50%. CT Analyzer software was employed for morphometric quantification of trabecular bone indices like morphology in culture, and their clonogenic potential was such as volumetric bone mineral density (BMD; g/cm3), trabecular confirmed by classical CFU-F assay (Fig. 1A). These cells were bone volume fraction (BV/TV %), trabecular number (Tb. N; 1/mm), strongly positive for surface expression of MSC markers CD90, http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. ASCs inhibit RANKL-induced osteoclast differentiation in vitro. M-CSF–dependent OCPs derived from bone marrow of DBA/1J mice were cultured in the presence of M-CSF (M, 30 ng/ml) and RANKL (R, 40 ng/ml) with or without ASCs. (A) OCPs (1 3 105 cells/well) were cultured in 48-well plates with graded ratios of ASCs, from 500:1 (OCP/ASC) to 10:1 and number of osteoclasts were counted as TRAP+ MNCs (three or more nuclei). The p values are with respect to M-CSF and RANKL group without ASCs. (B) Representative images of coculture at an OCP/ASC ratio of 50:1, stained for TRAP and counterstained with hematoxylin (original magnification 310). (C) Markers of osteoclast formation and function, namely TRAP, CTR, DC-STAMP, integrin b3, MMP9, and cathepsin K (Cat K), were measured at the mRNA level by real-time PCR. (D) RANK and c-fms expression was also measured by quantitative PCR. Results are representative of two independent experiments. ###p # 0.001 compared with M-CSF group; *p # 0.05, ***p # 0.001 compared with M-CSF and RANKL group. AM, M-CSF with ASCs; AMR, M-CSF and RANKL with ASCs; M, M-CSF; MR, M-CSF and RANKL. 5140 STEM CELL THERAPY IN MOUSE MODEL OF RHEUMATOID ARTHRITIS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. ASCs inhibit RANKL-induced osteoclast differentiation in a contact-independent manner. OCPs from mouse bone marrow were cultured in the presence of M-CSF and RANKL with or without ASCs in a contact-independent manner. (A) Representative photographs of TRAP+ MNCs of Transwell cultures of osteoclast differentiation with OCPs in the lower chamber and ASCs in culture inserts. Original magnification 310. Representative images (original magnification 310) (B) and number of osteoclasts (C) in cultures where OCPs (5 3 104 cells/well) were differentiated into osteoclasts in 96-well plates in the presence or absence of ASC-CM (S) for 3–4 d are shown. ASC-CM was added at two different concentrations (25% and 50%). Gene ex- pression analysis of markers of osteoclasts (D) and receptors of OCPs (E) is shown. Results are representative of three (A–C) and two (D and E) independent experiments. (F and G) OCPs were differentiated in 96-well plates coated with a calcium phosphate film, with or without 50% (Figure legend continues) The Journal of Immunology 5141 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. ASC-CM potently inhibits osteoclastogenesis. M-CSF–dependent OCPs were differentiated into osteoclasts using M-CSF (30 ng/ml) and RANKL (40 ng/ml). ASC-CM was added at three different time points along the course of differentiation, that is, on days 0, 1, and 2. Cultures were fixed after 4 d and stained for TRAP. Representative images (A) and numbers (B) of osteoclasts in the cultures are shown (original magnification 310). ###p # 0.001 compared with M-CSF group; ***p # 0.001 compared with M-CSF and RANKL group. (C and D) OCPs were cultured using M-CSF and RANKL (10 ng/ml) with or without proinflammatory cytokines TNF-a, IL-17, and IL-1b at a concentration of 30 ng/ml. ASC-CM was added to these cultures at 50% volume. Cultures were terminated after 3 d and stained for TRAP (original magnification 310). Numbers are represented as mean 6 SEM of five replicates in each group. Data are shown as representative images (C) and numbers of osteoclasts (D) of two independent experiments. M, M-CSF; MR, M-CSF and RANKL; MRS, M-CSF and RANKL with ASC-CM.

CD44, CD29, platelet-derived growth factor receptor, Sca-1, CD73, cycles of induction and maintenance, oil globules were formed and CD105 and were negative for the hematopoietic marker CD34 within cells confirmed by Oil Red O staining (Fig. 1D). For and monocyte/macrophage markers CD14 and CD11b. These cells chondrogenic differentiation, micromass pellets of ASCs were expressed MHC class I but not MHC class II, suggesting their cultured in the presence of chondrogenic media for 21 d. Histo- compatibility for allogenic transplantation (Fig. 1B). The multi- logical examination of H&E-stained sections of pellets showed lineage differentiation potential was determined by culturing ASCs differentiated chondrocytes present in the lacunae (Fig. 1E). To in lineage-specific conditions. ASCs formed mineralized nodules check the immunosuppressive potential of ASCs, we measured the in the presence of osteogenic factors, confirmed by Alizarin red proliferation of activated CD4+ T cells in the presence or absence S staining (Fig. 1C). Upon adipogenic induction for six or seven of ASCs or ASC-CM. For this, splenic CD4+ T cells (2.5 3 105

ASC-CM (S) for 8 d. Cells were washed off and resorption of the calcium phosphate film by osteoclasts was assessed by bright-field microscopy. Original magnification 310. Data are shown as representative images (F) and percentage resorption (G) of two independent experiments with eight replicates in each. ###p # 0.001 compared with M-CSF group; *p # 0.05, **p # 0.01, ***p # 0.001 compared with M-CSF and RANKL group. M, M-CSF; MR, M-CSF and RANKL; MRS, M-CSF and RANKL with ASC-CM; MS, M-CSF with ASC-CM. 5142 STEM CELL THERAPY IN MOUSE MODEL OF RHEUMATOID ARTHRITIS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. ASCs decrease the disease severity and bone destruction in CIA. CIA was induced in 8- to 10-wk-old DBA/1J mice as described in Materials and Methods.Onday22,23 106 ASCs in 100 ml PBS were injected i.p. to one group and an equal volume of PBS to the other group of CIA mice. Severity of arthritis was evaluated by measuring mean arthritic score of four limbs per animal (A) and hindpaw thickness (B) until day 36. Data are presented as mean 6 SEM (n = 6–7/ group). Significance was calculated by two-way ANOVA followed by a Bonferroni multiple comparisions test between 1) control versus CIA plus PBS groups and 2) CIA plus PBS versus CIA plus ASC groups. Similar results were obtained in four independent experiments. Representative images (C) and radiographs (D)ofhindpaws of mice on day 36 are shown. Yellow arrows in (D) show bone density at the metatarsophalangeal joints. (E) Sections of knee joints were stained with H&E and histologically evaluated for cellular infiltration and pannus formation (n = 4–6 mice/group). Original magnification 310. Significance was calculated by a Student t test between CIA plus PBS and CIA plus ASC groups. Similar results were obtained in three independent experiments. Hindlimbs along with ankle joints were dissected out and analyzed for histomorphometric measurements using mCT. (F) Representative three-dimensional images of distal tibiae from control (nonarthritic), PBS-treated, and ASC-treated CIA mice. (G) Trabecular bone indices, including volumetric bone mineral density (trabecular BMD [Tb. BMD]), BV/TV, Tb. Th, Tb. Sp, Tb. N, and Conn. D, were quantified from mCT reconstructions. Data are pooled from three independent experiments and are presented as mean 6 SD of 14–18 mice per group. Significance was calculated by a one-way ANOVA with a Bonferroni multiple comparisons test between 1) control versus CIA plus PBS groups and 2) CIA plus PBS versus CIA plus ASC groups. ##p # 0.01, ###p # 0.001 with respect to control group; *p # 0.05, **p # 0.01, ***p # 0.001 with respect to CIA plus PBS group. cells/ml) were cultured with either ASCs (2.5 3 103 cells/ml) or [3H]thymidine incorporation assay. ASCs (Fig. 1F) as well as ASC-CM (50% of culture volume) for 72 h along with T cell ASC-CM (Fig. 1G) inhibited proliferation of activated CD4+ Tcells activation Dynabeads. T cell proliferation was measured by a under in vitro conditions. These results indicate the clonogenic, The Journal of Immunology 5143 multilineage differentiation and immunosuppressive properties of OCPs were differentiated with M-CSF (30 ng/ml) and low con- isolated ASCs. ASCs of passages two or three were used in all centration of RANKL (10 ng/ml) along with 30 ng/ml TNF-a, further experiments. IL-17, or IL-1b. ASC-CM was added at 50% volume to these cultures. We observed that TNF-a strongly synergized with ASCs inhibit RANKL-induced osteoclastogenesis in vitro in RANKL and induced the formation of many giant osteoclasts. both a contact-dependent and -independent manner IL-17 and IL-1b also enhanced formation of multinuclear os- The effect of MSCs on osteoclast differentiation has not been well teoclasts. Interestingly, ASC-CM inhibited osteoclastogenesis studied in either mice or humans. To investigate the role of ASCs on in the presence of all the proinflammatory cytokines (Fig. 4C, in vitro osteoclastogenesis, we differentiated mouse bone marrow– 4D). These results indicate a potent inhibitory effect of ASCs on derived M-CSF–dependent OCPs into multinuclear osteoclasts, osteoclastogenesis. using M-CSF (30 ng/ml) and RANKL (40 ng/ml). ASCs were added to cultures in increasing ratios ranging from 200 to 10,000 ASCs reduce disease severity and bone destruction in CIA mice 3 5 cells per well, keeping numbers of OCPs (1 10 cells/well) Having demonstrated the potential of ASCs in inhibiting osteo- + constant. After 3 d, cells were stained for TRAP, and TRAP clastogenesis in the presence of proinflammatory cytokines, we multinuclear cells (MNCs) with three or more nuclei were counted wanted to validate this in an in vivo model of inflammation-induced as osteoclasts. We observed that ASCs significantly decreased the bone loss. The CIA model shares many pathological features with number of osteoclasts at a minimal OCP/ASC ratio of 100:1, with human RA, including synovial hyperplasia, joint swelling, bone complete absence of osteoclasts at an OCP/ASC ratio of 10:1 and cartilage destruction, and autoimmune T cell responses. CII, (Fig. 2A). M-CSF–treated OCPs without RANKL served as con- the main component of articular cartilage, is given as the auto- Downloaded from trol with no osteoclasts, and no deleterious effect on mononuclear antigen in this model (28). Genetically susceptible DBA/1J mice cells was seen in the presence of ASCs (Fig. 2B). The inhibition were immunized with CII emulsified in CFA on day 0, and an of osteoclast differentiation was further assessed by analyzing equal amount of CII emulsified in IFA was given as a booster dose markers associated with osteoclast differentiation and activation at on day 21. One day after the booster dose, mice received i.p. in- the transcript level. Osteoclast markers such as TRAP, CTR, and jections of 2 million murine syngeneic ASCs in 100 ml PBS or

DC-STAMP were significantly downregulated in cocultures with PBS alone. Mice of similar age group and gender were taken as http://www.jimmunol.org/ b ASCs (Fig. 2C). Expression of integrin 3, MMP9, and cathepsin normal controls. Mice were assessed for clinical symptoms every K was also decreased to some extent by ASCs. Notably, ASCs third day from day 21 until day 36 of the initial immunization. In alone did not induce osteoclast differentiation, as indicated by the control CIA mice, symptoms started to appear from day 24 and + absence of TRAP MNCs (Fig. 2B) and osteoclast markers reached a score of .10 per animal by day 36. However, mice (Fig. 2C) in cultures without RANKL. The receptor for RANKL, treated with ASCs developed a less severe form of arthritis as seen RANK, was also downregulated by ASCs, and c-fms, the receptor by decreased mean arthritic score (Fig. 5A) and hindpaw thickness for M-CSF, did not show any significant change in its expression (Fig. 5B). Images (Fig. 5C) of hindpaws of mice showed significant in the presence of ASCs (Fig. 2D). reduction in inflammation and soft tissue swelling, and radiographs To check whether the observed inhibitory effect of ASCs on (Fig. 5D) showed reduced bone erosions in the metatarsophalangeal by guest on September 27, 2021 osteoclastogenesis was cell–cell contact-dependent or -independent, joints upon treatment with ASCs. Mice were sacrificed on day 36 for we set up Transwell assays, where ASCs were separated from OCPs histopathological examination of joints. Reduced severity of arthritis m using 0.8- m culture inserts. ASCs were added in the upper chamber in ASC-treated mice was associated with decreased infiltration of and OCPs in the lower chamber along with M-CSF and RANKL for immune cells and pannus formation in affected joints (Fig. 5E). 3 d. ASCs retained the inhibitory effect on osteoclast differentiation, Histological analysis also revealed reduced subchondral bone erosions indicating the involvement of soluble mediators in the inhibition of in ASC-treated mice. To further evaluate the effect on local bone osteoclastogenesis (Fig. 3A). In support of this, ASC-CM also destruction, epiphyseal trabecular bone of distal tibia was subjected + inhibited the formation of TRAP MNCs. ASC-CM at 25% volume to mCT analysis. ASCs protected the collagen-immunized animals partially inhibited osteoclast differentiation whereas complete inhibi- from severe bone destruction as seen in representative mCT images tion was seen at 50% volume (Fig. 3B, 3C). The inhibition was further (Fig. 5F) and histomorphometric measurements (Fig. 5G). Loss of confirmed by downregulation of osteoclast markers at the mRNA level volumetric BMD, BV/TV, Tb. Th, and Tb. N in CIA mice was (Fig. 3D). ASC-CM did not affect the expression of RANK and c-fms significantly prevented in mice treated with ASCs (Fig. 5G). on OCPs as analyzed by real-time PCR (Fig. 3E). We also differen- Increased Tb. Sp in CIA mice was also slightly decreased upon tiated OCPs in the presence or absence of ASC-CM in 96-well plates treatment with ASCs (Fig. 5F). These data indicate that ASCs coated with a thin film of calcium phosphate for 8 d. No clear zones reduce clinical symptoms, joint pathology, and local bone de- of resorption were formed in the presence of ASC-CM, further struction in CIA mice. confirming inhibition of osteoclast formation (Fig. 3F, 3G). ASCs decrease periarticular and systemic osteopenia in CIA mice To determine the stage at which osteoclast differentiation was affected by ASC-CM, we cultured OCPs with M-CSF and RANKL, Inflammatory processes in RA result in adverse effects on bone and ASC-CM (50%) was added on days 0, 1, and 2. Cultures were remodeling, skewing the balance toward resorption. The effect of fixed and stained for TRAP on day 4. We observed complete in- ASCs on inflammation-induced periarticular and systemic bone hibition of osteoclast formation when ASC-CM was added on days loss was assessed by mCT of trabecular and cortical bone struc- 0 and 1, and significant inhibition was also seen when ASC-CM was tures. The distal femur and fifth lumbar vertebra (L5) were ana- added on day 2 (Fig. 4A, 4B). All these in vitro data clearly indicate lyzed for BMD and trabecular bone parameters. Additionally, a strong anti-osteoclastogenic potential of ASCs in vitro. cortical bone at femur mid-diaphysis was analyzed. Arthritis was Proinflammatory cytokines, such as TNF-a,IL-17,andIL-1, associated with an increase in trabecular bone loss in the meta- are present at the sites of inflammatory bone erosions, and they physeal region of both distal femur (Fig. 6A, 6B) and proximal act synergistically with RANKL in enhancing osteoclastogenesis tibia (data not shown). Representative images (Fig. 6A) and histo- (3, 26, 27). We further evaluated whether ASCs can inhibit osteo- morphometric measurements (Fig. 6B) of cancellous bone of distal clastogeneis in the presence of these proinflammatory cytokines. femora of control nonarthritic, PBS-treated, and ASC-treated CIA 5144 STEM CELL THERAPY IN MOUSE MODEL OF RHEUMATOID ARTHRITIS

FIGURE 6. ASCs protect from periarticular and systemic bone loss in CIA mice. CIA mice were injected i.p. with PBS or 2 3 106 ASCs on day 22 of primary immunization and sacri- ficed on day 36. (A) Representative three-di- mensional images of distal femur from control nonarthritic, PBS-treated, and ASC-treated CIA mice. (B) Trabecular bone indices, in- cluding Tb. BMD, BV/TV, Tb. Th, Tb. Sp, Tb. N, and Conn. D, were quantitated from mCT reconstructions. Data are pooled from three independent experiments and are presented as mean 6 SD of 14–18 mice per group. Repre- sentative two-dimensional images (C)and quantitative measurements (D)offemurmid- diaphysis Ct. BMD and Cs. Th are shown. Data are pooled from two independent experi- ments and are presented as mean 6 SD of 9–11 Downloaded from mice per group. Representative three-dimen- sional images (E) and trabecular parameters (F) of the fifth lumbar vertebra (L5) are shown. Data are pooled from three independent ex- periments and are presented as mean 6 SD of 14–18 mice per group. Significance was cal-

culated by a one-way ANOVA with a Bonfer- http://www.jimmunol.org/ roni multiple comparisons test between 1) control versus CIA plus PBS groups and 2) CIA plus PBS versus CIA plus ASC groups. #p # 0.05, ##p # 0.01, ###p # 0.001 with re- spect to control group; *p # 0.05, **p # 0.01, *** p # 0.001 with respect to CIA plus PBS group. by guest on September 27, 2021 mice showed protection of trabecular structure in ASC-treated mice of CIA mice was bigger compared with normal mice. Similarly, compared with PBS-treated arthritic controls. A significant increase the increased size of osteoclasts explains the decrease in their in trabecular bone indices, including trabecular BMD (Tb. BMD), numbers at a concentration of 50 ng/ml RANKL. Osteoclast BV/TV, Tb. N, and Conn. D, was observed in ASC-treated mice numbers were decreased in ASC-treated CIA mice compared with with respect to PBS-injected CIA mice. Increased Tb. Sp in CIA PBS-treated ones at both concentrations of RANKL (Fig. 7A, 7B). mice was significantly decreased by ASC treatment (Fig. 6B). A The decreased osteoclastogenesis in ASC-injected mice could be a similar pattern was observed in BV/TV, Tb. Sp, and parameters result of either decreased numbers of osteoclast precursors or RANK of trabecular microstructure of tibiae (data not shown). Femoral mid- expression on precursors, leading to a decreased sensitivity toward diaphyseal scanning showed no significant difference in Ct. BMD RANKL. The percentage of OCPs, indicated by the CD11b+c-fms+ and Cs. Th between the groups (Fig. 6C, 6D). We also performed population, was higher in PBS-treated CIA mice compared with mCT of L5 (mostly consisting of trabecular bone) to evaluate the normal controls, which was significantly decreased in CIA mice effect of ASC treatment on axial skeleton. Significant bone loss was treated with ASCs (Fig. 7C). However, no significant change was seen in CIA mice compared with normal controls, and treatment with observed in their RANK expression (Fig. 7D). Absence of significant ASCs significantly prevented this loss, indicated by increased changes in the percentages of F4/80+ macrophages (Supplemental BMD and improved trabecular parameters (Fig. 6E, 6F). All of these Fig. 1A), CD11c+ dendritic cells (Supplemental Fig. 1B), and c-Kit+ data confirm a strong protective effect of ASCs on inflammation- myeloid progenitors (Supplemental Fig. 1C) in all three groups of induced bone loss in CIA mice. mice implies lack of deleterious effect of ASCs on other myeloid populations of bone marrow. These results indicate that enhanced ASCs decrease osteoclastogenesis in arthritic mice by reducing osteoclastogenesis in CIA mice was reduced upon treatment with osteoclast precursors ASCs as a result of decreased osteoclast precursor pool. We next investigated the mechanisms underlying decreased se- verity of arthritis and bone loss upon treatment with ASCs. To study ASCs promote immune tolerance in CIA mice by reducing the levels of osteoclastogenesis in ASC-treated mice, bone mar- autoimmune T cell proliferation and enhancing row–derived stromal cell-free OCPs from all three groups of mice regulatory lymphocyte population were differentiated into osteoclasts using M-CSF and two different Autoimmune T and B cell responses against CII lead to systemic concentrations of RANKL (25 and 50 ng/ml) ex vivo. CIA mice inflammation, which subsequently drives bone destruction in CIA showed increased osteoclast numbers compared with control mice (28). However, a regulatory subset of T cells (Tregs) is involved in at a higher concentration of RANKL (50 ng/ml). Although there inducing tolerance and confers protection against CIA by preventing was no significant difference in the number of osteoclasts at a autoimmune responses (29) and bone loss (30). We observed that lower concentration of RANKL (25 ng/ml), the size of osteoclasts treatment of CIA mice with ASCs inhibited proliferation of dLN The Journal of Immunology 5145 Downloaded from http://www.jimmunol.org/

FIGURE 7. ASCs decrease RANKL-induced osteoclastogenesis in CIA mice by reducing osteoclast precursors. CIA mice were injected i.p. with PBS or 2 3 106 ASCs on day 22 of the first immunization and sacrificed on day 36. Bone marrow was harvested and M-CSF–dependent OCPs were differentiated into osteoclasts in the presence of M-CSF with or without two different concentrations of RANKL (25 or 50 ng/ml). Representative images (A) and numbers of osteoclasts (B) in cultures are shown as a measure of osteoclastogenesis (original magnification 310). (C) Percentage of osteoclast precursors (CD11b+c-fms+) in bone marrow. (D) The amount of RANK expressed on CD11b+ cells of bone marrow. Data are presented as mean 6 SEM of seven mice per group in (B), four to five mice per group in (C), and five mice per group in (D). Significance was calculated by a one-way ANOVA with a Bonferroni multiple comparisons test between 1) control versus CIA plus PBS groups and 2) CIA plus PBS versus CIA plus ASC groups. Similar results were obtained # # ### # # # # in three independent experiments. p 0.05, p 0.001 with respect to control group; *p 0.05, **p 0.01, ***p 0.001 with respect to CIA plus by guest on September 27, 2021 PBS group. M, M-CSF; MR, M-CSF and RANKL. cells in response to CII (40 ng/ml), compared with PBS treatment, osteopenia and leading to systemic bone loss. Al- as indicated by stimulation indices (Fig. 8A). Non–Ag-specific though reduction in inflammatory synovitis can be achieved by T cell proliferation measured in response to anti-CD3ε was also conventional antirheumatics, protection against structural damage significantly reduced upon treatment with ASCs compared with PBS requires complete abrogation of inflammation. This may lead to controls (Fig. 8A). This confirmed that ASCs markedly suppressed severe immune suppression posing a threat of infections to the autoimmune T cell responses in CIA mice. ASCs, when cocultured treated individuals. The uncoupling of inflammation and bone with CD4+ T cells for 5 d under activation conditions, induced erosion processes in few individuals (33) further complicates the regulatory phenotype in T cells (Fig. 8B). Percentages of Tregs in- treatment strategy, rendering anti-inflammatory agents ineffective dicatedbyCD4+Foxp3+ dual-positive population in peritoneum, in preventing the structural damage to bone. Therefore, the agents synovium, peripheral blood, and dLNs were significantly increased affecting both inflammatory and skeletal components of the disease upon ASC treatment, compared with PBS-treated arthritic mice. can be effective treatment strategies in such conditions. MSCs have However, the percentage of splenic Tregs was unaffected (Fig. 8C). emerged as a promising therapeutic candidate in various autoim- These results indicate immunosuppressive and tolerogenic potential mune and degenerative diseases, with significant improvement in of ASCs in CIA mice. B cell depletion inhibits CIA, suggesting the pilot scale clinical trials. Although the immunomodulation by involvement of B cells and autoantibodies in the pathogenesis of MSCs in RA is well studied, the effect on bone destruction asso- CIA (31). However, Bregs with the CD19+CD1dhiCD5+ phenotype ciated with disease has not been understood. In this study, we used a were shown to possess protective effect in autoimmune disease homogeneous population of ASCs having both differentiation and models (32). We found an increase in the percentage of the splenic immunosuppressive properties, and we investigated their effect on Breg subset in CIA mice upon treatment with ASCs (Fig. 8D). The bone loss along with autoimmune responses in CIA mice. serum levels of total anti-CII IgG Ab were unaffected in ASC- The role of osteoclast activation in systemic osteoporosis as- treated mice compared with PBS-treated animals (Fig. 8E). These sociated with inflammatory arthritis has been verified in various results suggest that ASCs promote immune tolerance by increasing animal models. Inhibiting osteoclast differentiation and function the number of regulatory lymphocytes in CIA mice. becomes an important part of the treatment regimen when bone destruction is the therapeutic target. We observed a strong inhib- Discussion itory potential of ASCs on RANKL-induced osteoclast differen- Human RA is a complex disease affecting multiple systems in the tiation in both a contact-dependent and -independent manner. body, predominantly inflammatory synovitis and structural damage Previously, murine MSCs derived from compact bone have been to bone. Also, RA is an independent risk factor for generalized shown to promote osteoclastogenesis in vitro through expression 5146 STEM CELL THERAPY IN MOUSE MODEL OF RHEUMATOID ARTHRITIS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 8. ASCs reduce CII-specific T cell proliferation and induce regulatory lymphocytes in arthritic mice. CIA mice were injected with PBS or 2 3 106 ASCs on day 22 of initial immunization. On day 36, mice were sacrificed and analyzed for immune parameters. (A) dLN cells were cultured in the presence of CII (40 mg/ml) or plate-bound anti-CD3ε (2 mg/ml) for 72 h and proliferation was measured by [3H]thymidine incorporation assay. Stimulation indices (S.I.) values are calculated using the formula: cpm in response to Ag/cpm in absence of Ag. The inset in (A) shows the representation of decreased size of dLNs in ASC-treated CIA mice compared with PBS-treated controls. (B) Magnetically sorted splenic CD4+ T cells (5 3 105 cells/well) from DBA/1J mice were cultured with or without ASCs (5 3 103 cells/well) in 48-well plates for 72 h. Cells were stained for Foxp3 and IL-17A and analyzed by flow cytometry. Plots are representative of two independent experiments. (C) Representative plots and average percentages of CD4+Foxp3+ Tregs in the peritoneal cavity, synovium, peripheral blood, dLNs, and spleens of control, arthritic, and ASC-treated arthritic mice. (D) Percentages of CD1dhiCD5+ Bregs from CD19-gated population of splenocytes. (E) Anti-CII IgG titers were measured in serum by ELISA. Data are presented as mean 6 SEM of six to seven mice per group in (A), five to seven mice per group in (C), three to six mice per group in (D), and four mice per group in (E). Significance was calculated by a one-way ANOVAwith a Bonferroni multiple comparisons test between 1) control versus CIA plus PBS groups and 2) CIA plus PBS versus CIA plus ASC groups. Similar results were observed in two independent experiments. ##p # 0.01, ###p # 0.001 with respect to control group; *p # 0.05, **p # 0.01, ***p # 0.001 with respect to CIA plus PBS group. The Journal of Immunology 5147 of RANKL (17). In our study, murine ASCs alone could not in- decreased in CIA mice upon treatment with ASCs. ASCs decreased duce osteoclast differentiation despite their expression of RANKL OCPs in bone marrow without affecting the percentage of other (data not shown). Based on our results and existing literature, the myeloid lineage cells, including F4/80+ mature macrophages, multilineage and T cell suppressive properties seem to be common CD11c+ dendritic cells, and CD11b2c-Kit+ myeloid precursors. among MSCs derived from all sources; however, there might be In addition to increased osteoclastogenesis, bone formation is few tissue-specific functions that can be attributed to the variations also compromised in arthritis at sites of focal bone erosion (4, 41). in their anti-osteoclastogenic effect. Our results are in agreement MSCs derived from mouse bone tend to lose their differentiation with two other reports demonstrating the inhibitory effect of hu- potential in the presence of proinflammatory cytokines (42). Also, man bone marrow MSCs on in vitro osteoclastogenesis mediated lack of repair of bone erosion by antirheumatic drugs is due to through osteoprotegerin, a decoy receptor for RANKL, or through active suppression of bone formation by proinflammatory cyto- surface expression of CD200 (18, 19). Although our ASCs con- kines (5). However, we observed an increased mineralization of ASCs stitutively expressed osteoprotegerin (data not shown), the inhib- in the presence of two major proinflammatory cytokines of arthritic itory effect of ASC-CM on osteoclast differentiation in the synovium, TNF-a and IL-17 (Supplemental Fig. 2). These results presence of high concentrations of RANKL (200 ng/ml) (data not suggest that ASCs are resistant to inhibitory action of these shown) rules out its complete involvement in the inhibition of cytokines on osteoblast differentiation and bone formation. osteoclastogenesis. The complete mechanisms of inhibitory action Tregs play a crucial role in controlling autoimmunity by in- of ASCs on osteoclast formation is not yet understood, but we ducing tolerance, and they prevent the development of chronic speculate that it could be the concerted effect of all pro- and anti- inflammatory and autoimmune diseases (43). ASCs decreased osteoclastogenic molecules secreted by ASCs. Proinflammatory T cell proliferation in CIA mice, measured in response to anti- Downloaded from cytokines, such as TNF-a, IL-17, and IL-1, are present at the CD3ε and CII, the main autoantigen in the model. ASCs also sites of inflammatory bone erosions and act synergistically with promoted Treg generation in peripheral tissues, including dLNs, RANKL in enhancing osteoclastogenesis and bone loss (3, 26). peritoneum, synovium, and peripheral blood. The decreased au- The inhibitory effect of ASC-CM on the RANKL-induced oste- toimmune responses could be due to the direct effect of ASCs on oclast formation in the presence of these proinflammatory cyto- T cell suppression or indirectly through increased Treg population.

kines suggests a strong protective effect against inflammation- Tregs are also shown to inhibit osteoclastogenesis and protect http://www.jimmunol.org/ mediated bone destruction. bone destruction in arthritis (30, 44). The increased percentage of In our study, a single injection of murine ASCs at the onset of Tregs in ASC-treated mice could also have contributed to de- disease significantly reduced the severity of disease in CIA mice. creased osteoclastogenesis and bone loss in ASC-treated CIA Murine bone marrow MSCs have shown contradictory results in mice. Thus, ASCs affected osteoclastogenesis and bone loss in reducing the disease severity in CIA model, despite inhibiting CIA mice both directly and indirectly. T cell proliferation in response to CII (34–37). However, mouse In addition to Tregs, we also observed an increase in the per- and human ASCs have been reported to ameliorate CIA by centage of CD19+CD1dhiCD5+ Bregs in the spleens of ASC- modulating T cell responses (15, 38). It appears that MSCs from treated CIA mice compared with PBS-treated controls. Bregs adipose tissue could be advantageous over those from bone mar- were shown to have a protective effect in autoimmune disease by guest on September 27, 2021 row for the treatment of arthritis, although it needs to be supported models of experimental autoimmune encephalomyelitis and CIA by more experimental evidences. (45, 46). Also, human bone marrow MSCs were shown to up- CIA induced loss of trabecular bones at appendicular and axial regulate Bregs in an experimental autoimmune encephalomyelitis sites as assessed by mCT, and treatment with ASCs protected ar- model (47). This suggests that Bregs also contribute to the pro- thritic bone loss. Trabecular parameters of L5 vertebrae were also tective effect of ASCs in RA. Thus, we demonstrate that ASCs significantly improved in ASC-treated mice compared with ar- inhibit pathological bone loss by suppressing osteoclastogenesis thritic mice. These results suggested that ASC treatment protected and improving immune tolerance in CIA mice. Our results suggest not only periarticular but also systemic bone loss. Trabecular bone that ASCs have the potential to serve as better therapeutic agents remodeling (turnover) units occur in much greater numbers due to for RA. high surface area/bone matrix volume configuration than the cortical bone, resulting in bone turnover rate at the trabecular sites Acknowledgments being much higher than at the cortical sites (39). Therefore, in- We thank Snehal R. Joshi for critically reading the manuscript. creased osteoclastogenesis under any pathological condition such as the one we observed with CIA is more likely to result in the loss Disclosures of trabecular bones over cortical bones as the remodeling event is The authors have no financial conflicts of interest. triggered by osteoclasts. In the present study, cortical bone in the CIA group was maintained at the level of control whereas the References trabecular bones suffered significant loss during the duration of 1. Firestein, G. S. 2003. Evolving concepts of rheumatoid arthritis. Nature 423: this study. It is possible that a longer duration of CIA than the 356–361. present 36 d could have induced cortical bone loss. In our study, 2. Teitelbaum, S. L. 2000. Bone resorption by osteoclasts. Science 289: 1504–1508. treatment of arthritic mice with one dose of ASCs improved the 3. McInnes, I. B., and G. Schett. 2007. Cytokines in the pathogenesis of rheumatoid arthritis. Nat. Rev. Immunol. 7: 429–442. trabecular bone parameters and mitigated bone loss. Based on the 4. Schett, G., and E. Gravallese. 2012. Bone erosion in rheumatoid arthritis: present data, it is reasonable to speculate that the extent of pro- mechanisms, diagnosis and treatment. Nat. Rev. Rheumatol. 8: 656–664. tection conferred against trabecular bone loss in CIA could be 5. Dimitroulas, T., S. N. Nikas, P. Trontzas, and G. D. Kitas. 2013. Biologic therapies and systemic bone loss in rheumatoid arthritis. Autoimmun. Rev. 12: further improved by increased dosage or frequency of adminis- 958–966. tration of ASCs. 6. Pittenger, M. F., A. M. Mackay, S. C. Beck, R. K. Jaiswal, R. Douglas, In agreement with another study (40), there was an increase in the J. D. Mosca, M. A. Moorman, D. W. Simonetti, S. Craig, and D. R. Marshak. number of osteoclast precursors and osteoclastogenesis in CIA mice 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284: 143–147. compared with normal controls. In the present study, the number of 7. Jiang, Y., B. N. Jahagirdar, R. L. Reinhardt, R. E. Schwartz, C. D. Keene, OCPs and the osteoclastogenic potential of bone marrow cells were X. R. Ortiz-Gonzalez, M. Reyes, T. Lenvik, T. Lund, M. Blackstad, et al. 2002. 5148 STEM CELL THERAPY IN MOUSE MODEL OF RHEUMATOID ARTHRITIS

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